This project is based on a detailed analysis of the enzyme 5'nucleotidase i in rat liver. This enzyme is cncentrated in the plasma membrane, but is also present in other cytopllsmic membranes. The orientation of the enzyme in the membranes is being determined by cytochemical procedures which have been developed for use with isolated cell fractions. The factors responsible for the asymmetric localization of the enzyme arebeing investigated. The enzyme has been shown to bind specifically to a small fraction of the shingomyelin of the liver. The effect of this association on enzymatic activity is to be investigated, and the properties of the bound sphingomyelin are to be determined. Th enzyme has been purified 2800 fold from rat liver homogenates. When the protein which is responsible for enzymatic activity has been identified unqeuivocally, the synthesis, intracellular transport and degradation of the enzyme will be investigated.